CN112662658A - Production of L-phenylpyruvic acid by immobilized recombinant escherichia coli using L-phenylalanine - Google Patents
Production of L-phenylpyruvic acid by immobilized recombinant escherichia coli using L-phenylalanine Download PDFInfo
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Abstract
The invention discloses immobilized recombinant escherichia coli for producing L-phenylpyruvic acid by utilizing L-phenylalanine, belonging to the technical field of biocatalysis. The invention adopts an adsorption-crosslinking method, fixes recombinant escherichia coli expressing L-amino acid deaminase PmiLAAD from proteus mirabilis in a composite carrier of diatomite-polyethyleneimine-glutaraldehyde, and is used for producing L-phenylpyruvic acid by L-phenylalanine after optimization. The recovery of the total enzyme activity of the obtained immobilized cells is more than 60 percent. Coli cells are used as a biocatalyst to catalyze L-phenylalanine to produce L-phenylpyruvic acid for biological preparation, the substrate concentration is 100g/L, the reaction time is 16h, and the conversion rate is over 60 percent. The immobilized cells are recycled and reacted for 8 batches repeatedly, the enzyme activity is not obviously reduced, and the substrate conversion rate of each batch is more than 50 percent.
Description
Technical Field
The invention relates to a method for producing L-phenylpyruvic acid by using L-phenylalanine through immobilized recombinant escherichia coli, belonging to the technical field of biological catalysis.
Background
Phenylpyruvic Acid (PPA) is a multifunctional organic acid and is widely used in pharmaceutical, food and chemical industries. The traditional chemical preparation method has serious environmental pollution and low yield of a microbial fermentation method, and for a biotransformation method, D-amino acid oxidase (D-AAO) is generally adopted to catalyze D-phenylalanine to synthesize PPA, and a toxic byproduct hydrogen peroxide is generated in the reaction process. Sp from Proteus sp L-amino acid deaminases (L-AADs) specifically catalyze the oxidative deamination of L-Phenylalanine (PLA) to synthesize PPA without the production of hydrogen peroxide and with PLA being less expensive than D-phenylalanine. Thus, the use of biotransformation is a promising approach.
The cell is treated by utilizing the immobilization technology, so that the stability of enzyme and the cell can be enhanced, the tolerance of the cell to an organic solvent and a mechanical shearing force is improved, the content is prevented from being released to a reaction system after the cell is broken, and the influence on a product purification process is reduced. At present, the research in the field of immobilized cell/enzyme catalysis is less at home and abroad, and the related research has the problems of low total enzyme activity recovery rate, high preparation cost of immobilized enzyme, unsatisfactory catalytic activity, poor stability, low substrate concentration, poor tolerance of organic solvent and the like, thereby seriously limiting the application of the immobilized biocatalyst in the production of alpha-keto acid.
Disclosure of Invention
[ problem ] to
The technical problem to be solved by the invention is to provide a method for converting L-phenylalanine into L-phenylpyruvic acid by immobilized recombinant escherichia coli.
[ solution ]
The invention provides a method for immobilizing recombinant escherichia coli and a method for converting L-phenylalanine into L-phenylpyruvic acid by utilizing the immobilized recombinant escherichia coli. The recombinant escherichia coli is L-amino acid deaminase PmiLAAD from proteus mirabilis, and the recombinant escherichia coli is preferably E.coli BL21-pET-28 a-PmiLAAD. The diatomite is pretreated by acid washing.
The method for immobilizing recombinant Escherichia coli comprises the following steps:
(1) culturing recombinant escherichia coli capable of expressing L-amino acid deaminase from proteus mirabilis, collecting wet thalli, and preparing bacterial suspension;
(2) soaking diatomite in 2M hydrochloric acid, washing with deionized water to neutrality, vacuum filtering, drying, and sieving;
(3) adding pretreated diatomite into the bacterial suspension, stirring, adding 10% polyethyleneimine aqueous solution, stirring for 1-2h, adding 25% glutaraldehyde aqueous solution, carrying out crosslinking reaction for 1-2h at room temperature, carrying out suction filtration, washing a filter cake obtained by suction filtration twice by using PBS (phosphate buffer solution) with pH of 8.0, and removing the buffer solution by suction filtration to obtain the immobilized recombinant escherichia coli cell containing the L-amino acid deaminase gene; the adding amount of the diatomite is 8-20g/L (preferably 16g/L) of the bacterial suspension, and the using amount of the 10% polyethyleneimine aqueous solution is 2-6% (preferably 3%) of the volume of the bacterial suspension; the 25% glutaraldehyde solution in water is used in an amount of 2-6% (preferably 3%) by volume of the bacterial suspension.
In one embodiment, in step (1), wet bacteria obtained by fermentation culture of recombinant escherichia coli are suspended by PBS buffer solution with ph8.0 to obtain bacterial suspension; the wet thallus content in the bacterial suspension is 20-150 g/L.
In one embodiment, in the step (2), the diatomite is soaked in 2M hydrochloric acid for 4 hours, washed to be neutral by deionized water, filtered, dried at 65 ℃ and sieved by a 40-mesh sieve.
In one embodiment, the stepsThe wet cells were obtained as follows in step (1): inoculating recombinant Escherichia coli into LB liquid medium containing kanamycin with final concentration of 0.1g/ml, culturing at 37 deg.C and 200rpm for 12-16h, transferring to new liquid medium containing kanamycin TB with final concentration of 0.1g/ml at an inoculum size of 2%, and culturing at 37 deg.C and 200rpm to OD600Adding IPTG with final concentration of 0.4mM, inducing at 25 deg.C for 14 hr, and centrifuging to obtain wet thallus cells.
The invention also provides a method for converting L-phenylalanine into L-phenylpyruvic acid by using the immobilized recombinant escherichia coli, which comprises the following steps: the recombinant Escherichia coli immobilized cell containing L-amino acid deaminase is used as a catalyst, L-phenylalanine is added as a substrate, PBS buffer solution with the pH value of 8 is used as reaction liquid to jointly form a reaction system, and the reaction system is reacted for 16h under the conditions of 20-40 ℃, 200-500rpm (preferably 35 ℃, 200rpm), so that the L-phenylpyruvic acid is obtained. The reaction solution can be replaced by conversion solution, and the composition of the reaction solution is as follows: NaCl 8g/L, KH2PO4 0.2g/L，KCl 0.2g/L，Na2HPO4·12H2O 2.9g/L。
Furthermore, the dosage of the catalyst is 20-100g/L of reaction system, and the concentration of the substrate is 100g/L of reaction system.
[ advantageous effects ]
The invention provides a method for immobilizing microbial cells of L-amino acid deaminase gene engineering bacteria, wherein the total enzyme activity recovery of the immobilized cells is more than 60 percent. Coli cells are used as a biocatalyst to catalyze L-phenylalanine to produce L-phenylpyruvic acid for biological preparation, the substrate concentration is 100g/L, the reaction time is 16h, and the conversion rate is over 60 percent. The immobilized cells are recycled and reacted for 8 batches repeatedly, the enzyme activity is not obviously reduced, and the substrate conversion rate of each batch is more than 50 percent. The reaction process of the invention does not need to add exogenous coenzyme, and has extremely high application value in the industrial production of the ketoacid.
Drawings
FIG. 1 shows a repeated batch of immobilized cells
FIG. 2 shows the form of immobilized cells after the preparation
FIG. 3 is a scanning electron micrograph of immobilized cells
Detailed Description
The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto:
the construction method of recombinant Escherichia coli E.coli BL21-pET-28a-PmiLAAD is disclosed in patent CN 110643585A: a process for preparing alpha-keto-beta-methyl-n-pentanoic acid from amino acid deaminase.
The enzyme activity is defined as: 1 enzyme activity unit is the amount of enzyme that can convert 1 micromole of substrate, or 1 micromole of the relevant group in the substrate, in 1 minute at 30 ℃ and pH 8.5.
The method for measuring the enzyme activity in the free cells comprises the following steps: 15mL of a solution having a concentration of about 40g L-1The L-phenylalanine solution (dissolved in Tris buffer at pH 8.0) was preheated at 30 ℃ for 10 min. Then, 0.5g of wet thallus is weighed into a 250mL conical flask, 14.5mL of buffer solution is added, the thallus is evenly mixed by blowing and sucking, then the preservative film is sealed and preheated for 10min (simultaneously preheated with the L-phenylalanine solution), then the preheated L-phenylalanine solution is quickly poured into the preheated thallus suspension, and the mixture is placed into a shaking table with the temperature of 30 ℃ and the rpm of 200 for reaction for 30min after the preservative film is sealed. After the reaction is finished, taking a proper amount of reaction liquid, quickly centrifuging and diluting, and detecting the L-phenylpyruvic acid by using high performance liquid chromatography.
The method for measuring the enzyme activity in the immobilized cells comprises the following steps: 15mL of a solution having a concentration of about 40g L-1The L-phenylalanine solution (dissolved in Tris buffer at pH 8.0) was preheated at 30 ℃ for 10 min. Then, 0.5g of immobilized thallus is weighed in a 250mL conical flask, 14.5mL of buffer solution is added, the thallus is evenly mixed by blowing and sucking, then the preservative film is sealed and preheated for 10min (simultaneously preheated with the L-phenylalanine solution), then the preheated L-phenylalanine solution is quickly poured into the preheated thallus suspension, and the mixture is placed in a shaking table with the temperature of 30 ℃ and the rpm of 200 for reaction for 30min after the preservative film is sealed. After the reaction is finished, taking a proper amount of reaction liquid, quickly centrifuging and diluting, and detecting the L-phenylpyruvic acid by using high performance liquid chromatography.
The high performance liquid chromatography detection method of the L-phenylpyruvic acid comprises the following steps: liquid chromatography column: aminex HPX-87H, 300X 7.8nm, mobile phase: dilute sulfuric acid solution, flow rate: 0.6mL/min, column temperature: 35 ℃, product peak time: 9min, detection wavelength: 210 nm.
Example 1: cultivation of recombinant Escherichia coli
Recombinant Escherichia coli E.coli BL21-pET-28a-PmiLAAD was inoculated into LB liquid medium containing kanamycin to a final concentration of 0.1g/ml, cultured at 37 ℃ and 200rpm for 12 to 16 hours, transferred to a new liquid medium containing kanamycin TB to a final concentration of 0.1g/ml in an inoculum size of 2% by volume, cultured at 37 ℃ and 200rpm to an OD of 0.6 to 0.8, added with IPTG to a final concentration of 0.4mM, induced at 5 ℃ for 14 hours, centrifuged, and the supernatant was removed to obtain wet cells.
Example 2: screening of immobilized Material
Different materials are adopted to carry out immobilization on the recombinant escherichia coli, and the conversion rate and the stability of immobilized cells are observed. The method comprises the following steps:
1: polyvinyl alcohol-sodium alginate (SA-PVA);
2: agar (Agar);
3: carrageenan (Carrageenan);
4: activated carbon-glutaraldehyde (AC-GA);
5: diatomaceous earth-polyethyleneimine-glutaraldehyde (DME-PEI-GA).
The preparation method comprises the following steps:
1: polyvinyl alcohol-sodium alginate
1.0g of wet thallus is suspended in 5mL of normal saline to form a bacterial suspension, the bacterial suspension is uniformly mixed with 15mL of sodium alginate-polyvinyl alcohol mixed solution, and in the obtained mixture, the final mass concentration of the sodium alginate is 30g/L, and the final mass concentration of the polyvinyl alcohol is 100 g/L. The resulting mixture was slowly dropped into 200mL of 3.0% CaCl using a syringe2In the solution, the solution was left to stand at room temperature for 8 hours to harden. Pour out CaCl2The immobilized cells were washed 3 times with the solution and physiological saline and stored in a refrigerator at 4 ℃ for further use.
2: agar-agar
1g of the wet cells were dissolved in 10mL of 15% agar solution (dissolved at 50 ℃ C.), and the mixture was poured into a sterile dish to coagulate it, which was then cut into small pieces of 0.5X 0.5cm, washed three times with distilled water, and stored at 4 ℃ C.
3: carrageenan
1g of the wet cells were dissolved in 10mL of a 3% (w/v) carrageenan solution (dissolved at 50 ℃ C.), and the mixture was poured into a sterile dish to coagulate it after stirring, and then cut into small pieces of 0.5X 0.5cm, washed three times with distilled water, and stored at 4 ℃ C.
4: activated carbon-ZIF-8-glutaraldehyde
(1) Suspending 1.0g of wet Escherichia coli in 20mL of physiological saline, adding 0.72g of Activated Carbon (AC), and stirring for 20 min;
(2) adding 0.12mL of 7.5mM GA solution, and stirring for 10 min;
(3) the prepared immobilized cells were recovered by filtration and stored at 4 ℃ until use.
5: diatomaceous earth-polyethyleneimine-glutaraldehyde.
(1) Adding 0.06g of diatomite into 20mL of physiological saline containing 1g of wet escherichia coli, and stirring for 0.5 h; pretreating diatomite: soaking diatomite in 2M hydrochloric acid for 4h, washing with deionized water to neutrality, performing suction filtration, oven drying in a 65 deg.C oven, and sieving with 40 mesh sieve;
(2) adding 0.8mL of PEI solution (10%, v/v), pre-adjusting to pH8.5 with 1M hydrochloric acid, stirring for 1h, adding 0.2mLGA solution (25%, v/v) while stirring, and stirring for 1h to obtain orange solution;
(3) filtering the diatomite-PEI-GA immobilized solution, collecting the immobilized cells, washing the immobilized cells in physiological saline for 3 times, and storing at 4 ℃ until use.
The immobilized cells prepared according to the 5 methods are respectively put into a reaction, a PBS buffer solution with the pH value of 8 is used as a reaction solution, the immobilized cells and a substrate form a reaction system together, the dosage of the immobilized cells is 50g/L of the reaction system, the concentration of the substrate is 100g/L of the reaction system, and the reaction conditions are as follows: the pH of the reaction solution is 8.0, the temperature is 35 ℃, the reaction time is 16h, and the rotating speed is 200 rpm. After the reaction was completed, the yield of L-phenylpyruvic acid was measured by high performance liquid chromatography, and the conversion rate (ratio of yield to substrate amount) was calculated, and as shown in Table 1, the best results were obtained with diatomaceous earth-polyethyleneimine-glutaraldehyde as the immobilized carrier.
TABLE 1 conversion of immobilized cells of different materials
Example 3: condition optimization of immobilized cells
According to the screening result of the immobilized material in the embodiment 2, the diatomite-polyethyleneimine-glutaraldehyde is finally selected to immobilize the cells, and the method is as follows:
suspending wet bacteria obtained by fermenting and culturing recombinant escherichia coli by using 10mL of PBS (phosphate buffer solution) with pH8.0 to obtain bacterial suspension, wherein the wet bacteria content in the bacterial suspension is 50 g/L; respectively adding 0.04g, 0.08g, 0.12g, 0.16g and 0.2g of pretreated diatomite into the bacterial suspension (soaking the diatomite in 2M hydrochloric acid for 4h, then washing the diatomite with deionized water to be neutral, then carrying out suction filtration, drying the diatomite in a 65 ℃ oven and sieving the diatomite with a 40-mesh sieve), stirring the diatomite with a magnetic stirrer at room temperature (25-30 ℃) and 400rpm for 0.5h, then adding 10% (v/v) of polyethyleneimine aqueous solution, pre-adjusting the polyethyleneimine aqueous solution to pH8.5 with 1M hydrochloric acid, stirring the polyethyleneimine aqueous solution for 1h, finally adding 0.2mL of 25% glutaraldehyde aqueous solution, carrying out crosslinking reaction at room temperature for 1h, carrying out suction filtration, washing a filter cake twice with PBS buffer solution with pH8.0, and then carrying out suction filtration to remove the buffer solution, thus obtaining immobilized cells.
And then, measuring the enzyme activity of the immobilized cells by taking the enzyme activity of the free cells as 100%, and respectively measuring the enzyme activity of the immobilized cells under different diatomite addition amounts, wherein the ratio of the enzyme activity to the enzyme activity of the free cells is the relative enzyme activity. As shown in Table 2, the amount of diatomaceous earth added is preferably 16 g/L.
TABLE 2 Effect of different amounts of diatomaceous earth added on the relative enzyme activity of immobilized cells
Example 4: production of L-phenylpyruvic acid by immobilized cells
Suspending wet bacteria obtained by fermenting and culturing recombinant escherichia coli by using 10mL of PBS (phosphate buffer solution) with pH8.0 to obtain bacterial suspension, wherein the wet bacteria content in the bacterial suspension is 50 g/L; adding 0.16g of acid pretreated diatomite into the bacterial suspension (soaking the diatomite in 2M hydrochloric acid for 4h, washing the diatomite to be neutral by deionized water, performing suction filtration, drying the diatomite in a 65 ℃ oven, sieving the diatomite by a 40-mesh sieve), stirring the diatomite for 0.5h by a magnetic stirrer at room temperature (25-30 ℃) and 400rpm, adding 10% (v/v) of polyethyleneimine aqueous solution, pre-adjusting the polyethyleneimine aqueous solution to pH8.5 by 1M hydrochloric acid, stirring the mixture for 1h, finally adding 0.2mL of 25% glutaraldehyde aqueous solution, performing crosslinking reaction for 1h at room temperature, performing suction filtration, washing a filter cake twice by PBS (phosphate buffer solution) with the pH8.0, and performing suction filtration to remove the buffer solution to obtain immobilized cells. The photograph of the immobilized cells is shown in FIG. 2, and the SEM image is shown in FIG. 3.
50g/L of immobilized bacteria and 100g/L of substrate were suspended in 20ml of transformation solution (transformation solution composition: 8g/L NaCl, KH)2PO4 0.2g/L，KCl 0.2g/L，Na2HPO4·12H2O2.9 g/L) is carried out, the pH of reaction liquid is 8.0, the temperature is 35 ℃, the reaction time is 24h, and the rotating speed is 200 rpm. And centrifuging part of reaction liquid after 24 hours, filtering the supernatant obtained by centrifuging, detecting the content of the L-phenylpyruvic acid by using a high performance liquid chromatograph, and calculating the conversion rate of the immobilized cells to the L-phenylalanine. After the first reaction, the immobilized cells were recovered by suction filtration, and the next reaction was carried out by repeating 10 reactions, the results of which are shown in FIG. 1.
Comparative example 1 without pretreating diatomaceous earth
Suspending wet bacteria obtained by fermenting and culturing recombinant escherichia coli by using 10mL of PBS (phosphate buffer solution) with pH8.0 to obtain bacterial suspension, wherein the wet bacteria content in the bacterial suspension is 50 g/L; respectively adding 0.16g of unpretreated diatomite into the bacterial suspensions, stirring for 0.5h at room temperature (25-30 ℃) by using a magnetic stirrer at 400rpm, adding 10% (v/v) of polyethyleneimine aqueous solution, pre-adjusting to pH8.5 by using 1M hydrochloric acid, stirring for 1h, finally adding 0.2mL of 25% glutaraldehyde aqueous solution, carrying out crosslinking reaction for 1h at room temperature, carrying out suction filtration, washing filter cakes twice by using PBS buffer solution with pH8.0, and carrying out suction filtration to remove the buffer solution, thus obtaining immobilized cells.
50g/L of immobilized bacteria and 100g/L of substrate were suspended in 20ml of transformation solution (transformation solution composition: 8g/L NaCl, KH)2PO4 0.2g/L，KCl 0.2g/L，Na2HPO4·12H2O2.9 g/L) is carried out, the pH of reaction liquid is 8.0, the temperature is 35 ℃, the reaction time is 24h, and the rotating speed is 200 rpm. And centrifuging part of reaction liquid after 24 hours, filtering the supernatant obtained by centrifuging, detecting the content of the L-phenylpyruvic acid by using a high performance liquid chromatograph, and calculating the conversion rate of the immobilized cells to the L-phenylalanine. The conversion of the non-pretreated diatomaceous earth immobilized cells was found to be 41.21% and the conversion of pretreated diatomaceous earth was found to be 68.35%.
Comparative example 2 No pH adjustment treatment was performed on polyethyleneimine
Suspending wet bacteria obtained by fermenting and culturing recombinant escherichia coli by using 10mL of PBS (phosphate buffer solution) with pH8.0 to obtain bacterial suspension, wherein the wet bacteria content in the bacterial suspension is 50 g/L; respectively adding 0.16g of unpretreated diatomite into the bacterial suspension, stirring for 0.5h by using a magnetic stirrer at room temperature (25-30 ℃) under the condition of 400rpm, then adding 10% (v/v) of polyethyleneimine aqueous solution, stirring for 1h, finally adding 0.2mL of 25% glutaraldehyde aqueous solution, carrying out crosslinking reaction for 1h at room temperature, carrying out suction filtration, washing a filter cake twice by using PBS (phosphate buffer solution) with pH8.0, and then carrying out suction filtration to remove the buffer solution, thus obtaining immobilized cells.
50g/L of immobilized bacteria and 100g/L of substrate were suspended in 20ml of transformation solution (transformation solution composition: 8g/L NaCl, KH)2PO4 0.2g/L，KCl 0.2g/L，Na2HPO4·12H2O2.9 g/L) is carried out, the pH of reaction liquid is 8.0, the temperature is 35 ℃, the reaction time is 24h, and the rotating speed is 200 rpm. And centrifuging part of reaction liquid after 24 hours, filtering the supernatant obtained by centrifuging, detecting the content of the L-phenylpyruvic acid by using a high performance liquid chromatograph, and calculating the conversion rate of the immobilized cells to the L-phenylalanine.
Polyethyleneimine was used for preparation of immobilized cells without pH treatment (pH 13), and the immobilized cell conversion rate was found to be 45.15%, which was decreased by 23.2% compared to the immobilized cell conversion rate after pH treatment (68.35%).
Claims (9)
1. The method for immobilizing recombinant escherichia coli is characterized in that the recombinant escherichia coli is immobilized in a diatomite-polyethyleneimine-glutaraldehyde composite carrier and used for catalyzing L-phenylalanine production to obtain L-phenylpyruvic acid, the recombinant escherichia coli expresses L-amino acid deaminase PmiLAAD from proteus mirabilis, and the diatomite is subjected to acid washing pretreatment.
2. The method for immobilizing recombinant Escherichia coli according to claim 1, comprising the steps of:
(1) culturing recombinant escherichia coli capable of expressing L-amino acid deaminase from proteus mirabilis, collecting wet thalli, and preparing bacterial suspension;
(2) soaking diatomite in 2M hydrochloric acid, washing with deionized water to neutrality, vacuum filtering, drying, and sieving;
(3) adding diatomite pretreated by acid into the bacterial suspension, stirring, adding 10% polyethyleneimine aqueous solution, stirring for 1-2h, adding 25% glutaraldehyde aqueous solution, carrying out crosslinking reaction for 1-2h at room temperature, carrying out suction filtration, washing a filter cake obtained by suction filtration twice by using PBS (phosphate buffer solution) with pH8.0, and removing the buffer solution by suction filtration to obtain the immobilized recombinant escherichia coli cell containing the L-amino acid deaminase gene; the adding amount of the diatomite is 8-20g/L of the bacterial suspension, and the using amount of the 10% polyethyleneimine water solution is 2-6% of the volume of the bacterial suspension; the dosage of the 25% glutaraldehyde aqueous solution is 2-6% by volume of the bacterial suspension.
3. The method for immobilizing recombinant Escherichia coli as claimed in claim 2, wherein in step (1), wet bacteria obtained by fermentation culture of recombinant Escherichia coli are suspended in PBS buffer solution with pH8.0 to obtain bacterial suspension; the wet thallus content in the bacterial suspension is 20-150 g/L.
4. The method for immobilizing recombinant escherichia coli as claimed in claim 2, wherein in the step (2), the diatomite is soaked in the 2M hydrochloric acid for 4 hours, washed to be neutral by deionized water, filtered, dried at 65 ℃ and sieved by a 40-mesh sieve.
5. The method of claim 2, wherein the wet biomass is obtained by the following steps: inoculating recombinant Escherichia coli into LB liquid medium containing kanamycin with final concentration of 0.1g/ml, culturing at 37 deg.C and 200rpm for 12-16h, transferring to new liquid medium containing kanamycin TB with final concentration of 0.1g/ml at an inoculum size of 2%, and culturing at 37 deg.C and 200rpm to OD600Adding IPTG with final concentration of 0.4mM, inducing at 25 deg.C for 14 hr, and centrifuging to obtain wet thallus cells.
6. Immobilized recombinant E.coli obtainable by the process according to any one of claims 1 to 5.
7. A method for converting L-phenylalanine to L-phenylpyruvic acid using the immobilized recombinant Escherichia coli of claim 6, comprising the steps of: the recombinant Escherichia coli immobilized cell containing L-amino acid deaminase is used as a catalyst, L-phenylalanine is added as a substrate, PBS buffer solution is used as reaction liquid to jointly form a reaction system, and the reaction is carried out at the temperature of 20-40 ℃ and the speed of 200-500rpm to obtain the L-phenylpyruvic acid.
8. The method according to claim 7, wherein the reaction solution is replaced with a conversion solution having the composition: NaCl 8g/L, KH2PO4 0.2g/L，KCl 0.2g/L，Na2HPO4·12H2O 2.9g/L。
9. The method of claim 7, wherein the amount of the catalyst is 20-100g/L and the concentration of the substrate is 100 g/L.
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| CN114258909B (en) * | 2021-12-14 | 2023-09-22 | 苏州良辰生物医药科技有限公司 | Cell fixing agent and cell fixing method |
| CN114686539A (en) * | 2022-04-29 | 2022-07-01 | 西北工业大学 | Method for preparing 5-methylpyrazine-2-carboxylic acid by using immobilized escherichia coli cells |
| CN115636985A (en) * | 2022-09-08 | 2023-01-24 | 广西大学 | Preparation method and application of antibacterial breathable biomass-based packaging film |
| CN115636985B (en) * | 2022-09-08 | 2023-08-04 | 广西大学 | Preparation method and application of antibacterial breathable biomass-based packaging film |
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